Multiple myeloma (MM) remains largely an incurable disease. The persistence of a drug-resistant cell population, probably the MM stem cells may be the cause of disease relapse. CD138-negative (CD138-) MM cell fraction was reported to contain MM stem cells. However, little is known about the molecular base of MM stem cells, which makes it difficult to specifically target these cells. Our preliminary data show that RAR1 is the top one up-regulated gene in CD138- MM stem cells compared to CD138+ tumor cells. RAR1 has two major isoforms, RARa1 and RARa2. Real-time PCR further demonstrated that it was RARa2 but not RARa1 that increased in CD138- MM stem cells. While RARa1 was ubiquitously expressed in MM cells, RARa2 was present in MM cells from about 30% of newly diagnosed patients. Patients with RARa2 expression had shorter overall-survival. RARa2-knockdown in RARa2+ MM cells induced cell growth arrest and apoptosis. Interestingly, ATRA selectively killed RARa2-overexpressing CD138- MM stem cells while sparing CD138+ tumor cells. Forced expression of RARa2 in RARa2-deficient MM tumor cells restored sensitivity to ATRA. We also found that Wnt signaling is activated in CD138- MM stem cells and ATRA down-regulates Wnt signaling. Our hypothesis is that RARa2 maintains the "stemness" and drug-resistance of MM stem cells by activating the Wnt/2-catenin pathway;and ATRA and other retinoids work through interference with Wnt signaling, which leads to loss of "stemness" features. The goal of this work is to determine the mechanisms by which RAR12 maintains the "stemness" features of MM stem cells and how retinoids kill MM stem cells. In specific Aim 1, we will clarify the mechanisms by which RARa2 maintains MM stem cell growth and survival. In specific Aim 2, we will clarify the role of Wnt/2-catenin pathway in ATRA-induced MM stem cell growth and survival. Finally, in specific Aim 3, we will exploit new approaches based on retinoid agents and drugs targeting Wnt pathway for the therapy of MM stem cells and MM disease in SCID-hu and 5TGM1 murine MM models. Our proposed study may provide better understanding of the biological functions of RARa2 in MM stem cells and is likely to add valuable information on new therapeutic approaches for MM stem cells which may significantly impact the clinical outcome of MM diseases. Since cancer stem cells exist in variant cancer malignancies, the proposed study may also have a major impact on the treatment of other cancers. PUBLIC HEALTH RELEVANCE: Cancer stem cells are identified in an increasing list of malignancies, including multiple myeloma, and are thought to be responsible for treatment failure and regrowth of cancers after treatment. However, the mechanisms by which cancer stem cells maintain their "stemness" features are still poorly understood. We have identified a protein in myeloma stem cells that may be responsible for stem cell growth and survival and may also play a crucial role in retinoid-based therapy in myeloma disease.